The present invention relates to the area of optical microscopy, more specifically, the area of confocal fluorescence microscopy, and methods of obtaining images of fluorescently labelled targets by confocal fluorescence microscopy.
Generally, most fluorescence microscopes can be categorized into one of the following general classes based on how the fluorescent image information is captured and processed:
Wide-field Microscopes
In these microscopes, the target is imaged using a conventional wide-field strategy as in any standard microscope, and collecting the fluorescence emission. Generally, the fluorescent-stained or labelled sample is illuminated with excitation light of the appropriate wavelength(s) and the emission light is used to obtain the image; optical filters and/or dichroic mirrors are used to separate the excitation and emission light.
Structured Light Microscopes
This is a modification of a microscope that provides an enhanced spatial resolution along an optical axis of the microscope. This feature allows for optical sectioning of the imaging specimen. The major component of the structured light illumination device is a one-dimensional optical grid. The grid pattern is systematically projected onto the specimen and is moved in the focal plane of the objective across the sample; the emission light is collected and compiled to create the image. One such “structured-light” image is generated after processing of three images of the specimen captured for different positions on the grid.
Ordinarily, the projected image of the grid provides a strong spatial modulation of the intensity for the object in the focal plane of the objective while the intensity modulation in the area above and below the focal plane is significantly reduced. As the emission radiation is collected, the image processing algorithm eliminates the weaker signals from above or below the primary image plane as defined by the grid. The resulting image is, thus, free of any stray light or out of focus data. Further, because the structured light illumination device utilizes the illumination source of the host wide-field microscope, all fluorescence capabilities of that microscope are maintained.
True Confocal Microscopes
Confocal microscopes utilize specialized optical systems for imaging. In the simplest system, a laser operating at the excitation wavelength of the relevant fluorophore is focused to a point on the sample; simultaneously, the fluorescent emission from this illumination point is imaged onto a small-area detector. Any light emitted from all other areas of the sample is rejected by a small pinhole located in front to the detector which transmits on that light which originates from the illumination spot. The excitation spot and detector are scanned across the sample in a raster pattern to form a complete image. There are a variety of strategies to improve and optimize speed and throughput which are well known to those skilled in this area of art.
Line-confocal Microscopes
This is a modification of the confocal microscope, wherein the fluorescence excitation source is a laser beam; however, the beam is focused onto a narrow line on the sample, rather than a single point. The fluorescence emission is then imaged on the optical detector through the slit which acts as the spatial filter. Light emitted from any other areas of the sample remains out-of-focus and as a result is blocked by the slit. To form a two-dimensional image the line is scanned across the sample while simultaneously reading the line camera. This system can be expanded to use several lasers and several cameras simultaneously by using an appropriate optical arrangement.
However, such line confocal microscopes are typically complex and costly, and thus not practical for many applications. There exists a real need for microscopes which combine the advantages of confocal and line confocal microscopes with the simplicity and economy of other systems.